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Publication Detail
Abnormal secretion and function of recombinant human factor VII as the result of modification to a calcium binding site caused by a 15-base pair insertion in the F7 gene
  • Publication Type:
    Journal article
  • Publication Sub Type:
    Article
  • Authors:
    Peyvandi F, Carew JA, Perry DJ, Hunault M, Khanduri U, Perkins SJ, Mannucci PM, Bauer KA
  • Publication date:
    2001
  • Pagination:
    960, 965
  • Journal:
    BLOOD
  • Volume:
    97
  • Issue:
    4
  • Print ISSN:
    0006-4971
  • Keywords:
    abnormal, ACID, activity, American, analysis, ANTIGEN, As, Aspartic Acid, BINDING, BINDING-SITE, biosynthesis, Blood, BLOOD-COAGULATION, BOND, calcium, catalytic, Catalytic Domain, cell, CELLS, COAGULATION-FACTOR VIIA, COMBINATION, COMPARTMENT, copy, DEFECT, DEFECTS, deficiency, degradation, DNA, DNA analysis, domain, duplication, ENDOPLASMIC-RETICULUM, English, expression, Factor VII, FACTOR-IX, FACTOR-VII, folding, formation, function, functional, GENE, Glutamic Acid, Graphic, Hematology, HEMOPHILIA, Histidine, homozygous, Hydrogen, Hydrogen bond, HYDROGEN-BOND, IDENTIFICATION, in vitro, In-vitro, INHIBITOR, INHIBITORS, INSERTION, Italy, LESS, Leucine, LEVEL, LOOP, loss, M, MECHANISM, media, modification, Molecular, Mutant, Mutation, novel, Nucleotide, PROCOAGULANT ACTIVITY, proteasome, PROTEIN, Protein Degradation, Protein Folding, recombinant, Result, secretion, Serine, SITE, small, societies, SOCIETY, SURFACE, Thrombosis, tissue factor, USA, VITRO, WILD-TYPE
  • Notes:
    Journal English Article AMER SOC HEMATOLOGY FEB 15 400HY WASHINGTON BLOOD 1900 M STREET. NW SUITE 200, WASHINGTON, DC 20036 USA
Abstract
A case of a novel mutation in the F7 gene that results in factor VII coagulant activity (VII:c) of less than 1% and VII antigen (VII:Ag) levels of 10% is presented. DNA analysis revealed a homozygous 15-base pair (bp) in-frame insertion-type mutation at nucleotide 10554, This insertion consisted of a duplication of residues leucine (L)213 to aspartic acid (D)217 (leucine, serine, glutamic acid, histidine, and aspartic acid), probably arising by slipped mispairing between 2 copies of a direct repeat (GCGAGCACGAC) separated by 4 bp, Molecular graphic analyses showed that the insertion is located at the surface of the catalytic domain in an exposed loop stabilized by extensive salt-bridge and hydrogen bond formation at which the calcium binding site is located. The mutation probably interferes with protein folding during VII biosynthesis and/or diminishes functional activity through the loss of calcium binding, In vitro expression studies demonstrated that the levels of VII:Ag in lysates of cells transfected with wild type VII (VIIWT) were equivalent to those with mutant type VII (VIIMT), but the level of secreted VIIMT was 5% to 10% that of VIIWT. Pulse chase studies demonstrated that VIIMT did not accumulate intracellularly, and studies with inhibitors of protein degradation showed that recombinant VIIMT was partially degraded in the pre-Golgi compartment, Accordingly, only small amounts of VIIMT with undetectable procoagulant activity were secreted into conditioned media. These results demonstrate that a combination of secretion and functional defects is the mechanism whereby this insertion causes VII deficiency. (C) 2001 by The American Society of Hematology
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